The present invention relates to the field of plant genetics. In particular, the invention provides novel selectable markers and promoters for plants.
The selection of mutants using cultured plant cells is in principle similar to that done with microorganisms, but in practice is much more difficult. The reasons for the difficulties include the usual clumpy nature of plant cell cultures; single cells or protoplasts usually cannot be easily grown to form clones, cell growth is slow and the cells are usually not monoploid. Despite these problems a large number of successful selection experiments have been carried out to produce mutants of value for producing compounds, for biochemical and molecular biology studies, for markers in genetic experiments and for improving crop plants. Part of the reason for the success is that cell systems allow the screening of millions of cells for the desired trait.
Whether the selected phenotype is under genetic or epigenetic control can most easily be determined by regenerating plants and by following the phenotype in progeny. Genetically controlled phenotypes would be inherited by progeny and would generally be more stable at the cell level in comparison to epigenetically controlled traits. A large number of in vitro selected traits have been shown to be expressed in regenerated plants and to be passed on to progeny.
There are several types of in vitro selection that can be used to obtain cells containing the trait of interest (J. Widholm, Iowa State J. of Research, 62: 587-597, 1988). These include selection for growth, selection for valuable compound production, auxotroph selection and resistance selection. Selection for resistance should be the easiest kind of selection to accomplish and from the number of reports in the literature this would appear to be true.
The selection for amino acid analog resistance in plants has been pursued for a number of years. A primary focus of this research has been directed to the enzyme anthranilate synthase (AS). AS catalyzes the conversion of chorismate into anthranilate, the first reaction leading from the common aromatic amino acid (shikimate),pathway toward the biosynthesis of tryptophan (Trp). As a branchpoint enzyme in the synthesis of aromatic amino acids, AS plays a key role in the diversion of chorismate into Trp and indolic secondary compound biosynthesis.
Available information indicates that AS plays a key role in regulation of Trp biosynthesis. In plants, bacteria, and fungi, AS activity is regulated by Trp feedback inhibition (Matsui et al., J. Bacteriol, 169: 5330-5332, 1987). In microbes, AS usually consists of two nonidentical subunits, referred to as the alpha subunit (component I) and the beta subunit (component II). component I can convert chorismate to anthranilate in the presence of high levels of ammonia (ammonia-dependent AS activity), whereas-component II is responsible for the use of glutamine (hereinafter referred to as xe2x80x9cGlnxe2x80x9d) as the amino donor (Hutter et al., Annu Rev Microbiol, 40: 55-77, 1986).
As a means to investigate regulation of the Trp pathway, toxic analogs of Trp have been used in metabolic studies of plant cell cultures and as a tool to select mutants. Many of these studies have been conducted with the growth inhibitor 5-methyltryptophan (5MT). In a number of species including Datura innoxia (hereinafter referred to as D. innoxia), Catharanthus roseus, and Solanum tuberosum, variant cell lines resistant to inhibitory concentrations of 5MT were found to have AS that was less sensitive to feedback inhibition by Trp (Carlson and Widholm, Physiol Plant, 44: 251-255, 1978; Scott et al., Phytochemistry, 18: 795-798, 1979; Ranch et al., Plant Physiol, 71: 136-140, 1983). Widholm (Planta, 134: 103-108, 1977) described 5MT-resistant carrot cell lines and a potato cell line that were auxin autotrophic.
In addition, 5-methylanthranilate was successfully used to isolate plant auxotrophic mutants defective in three different genes, trp1, trp2, and trp3 (Last and Fink, Science, 240: 305-310, 1988; Last et al., Plant Cell, 3: 345-358, 1991) and mutants of Chlamydomonas reinhardtii (Dutcher et al., Genetics, 131: 593-607, 1992). Mutants resistant to 5MT or alpha-methyltryptophan (xcex1MT) were reported in Arabidopsis thaliana (hereinafter referred to as A. thaliana) (Koornneef and van Loenen Martinet, Arabidopsis Inf Serv, 20: 104-108, 1983; Kreps and Town, Plant Physiol, 99: 269-275, 1992), maize (Kang and Kameya, Euphytica, 69: 95-101, 1993), Lemna gibba (Tam et al., Plant Physiol, 107: 77-85, 1995) and Oryza sativa (Lee and Kameya, Theor Appl Genet, 82: 405-408, 1991). The specificity of selection with these analogs have not been systematically investigated.
A feedback-insensitive AS gene (ASA1 mutant) has been recently obtained by selection of mutagenized Arabidopsis seeds resistant to 6-methylanthranilate (Li and Last, Plant Physiol., 110: 51-59, 1996). In addition, xcex1MT resistance led to identification of a mutant in A. thaliana with the same amino acid change (Kreps et al., Plant Physiol., 110: 1159-1165, 1996).
One method for the production of trahsgenic plants is to transform plant cells in tissue culture with a plasmid containing a promoter and selectable marker which also contains a gene (xe2x80x9cdesirable genexe2x80x9d) which would express the desired trait in the regenerated plant. Thus when one selects cells transformed with the selectable marker, many of these cells will also carry the desirable gene that will also be expressed to produce the desired result such as insect resistance, disease resistance, herbicide resistance, changed. starch, drought tolerance, etc. An example is where the nptII (neo) gene is driven by a constitutive promoter, nosP (Vermeulen et al., Plant. Cell Reports, 11: 243-247, 1992). Next to this selectable marker gene is a mutant acetolactate synthase gene with its own promoter. This latter gene makes the regenerated plants resistant to certain herbicides.
An AS gene which encodes an enzyme that is highly resistant to an amino acid analog, such as 5MT, would be an ideal selectable marker for the production of transgenic plants as described above. Especially if the promoter which regulates the expression of this enzyme provided for high level expression of the enzyme in tissue culture, and little or no expression in regenerated plants. There has been considerable environmental concern because most selectable markers are constitutively expressed in all tissues of the plant and are not of plant origin. The former concern would be reduced by using such a tissue culture specific promoter while the latter concern would be eliminated by using the plant-derived AS gene as the selectable marker. In fact, the use of a tissue culture specific promoter would even allow one to use selectable markers that are not of plant origin. Traditional selectable markers that are not of plant origin include nptII, which encodes kanamycin resistance.
A first aspect of the present invention is an isolated deoxyribonucleic acid (DNA) molecule comprising a DNA sequence (SEQ ID NO: 4), the ASA2 gene of Nicotiana tabacum (hereinafter referred to as N. tabacum), and fragments thereof, which encode a feedback-insensitive form of AS. The ASA2 gene product could function as a selectable marker for transforming plant cells.
A second aspect of the present invention is an isolated DNA molecule comprising a DNA promoter sequence, the ASA2 promoter sequence (SEQ ID NO: 14), which is capable of directing tissue culture specific transcription of a downstream structural gene in a plant cell. The functional promoter sequence may be selected from the group consisting of the tobacco ASA2 promoter and DNA sequences which are at least 70 percent homologous to a fragment of the tobacco ASA2 promoter which is from about 150 to about 606, more preferably from about 150 to about 370, and most preferably about 150 bases in length. For constitutive expression of the promoter, the fragment is preferably a fragment taken from between about xe2x88x92606 to about xe2x88x921 of the nucleotide sequence of the ASA2 promoter. For a functional promoter, the fragment preferably includes the xe2x88x92151 to xe2x88x92214 nucleotide sequence of the ASA2 promoter.
The tissue culture specific expression promoter sequence may be selected from the group consisting of the tobacco ASA2 promoter and DNA sequences which are at least 70 percent homologous to a fragment of the tobacco ASA2 promoter capable of directing tissue culture specific expression. The fragment is preferably between about 30 to about 100, more preferably 5 between about 30 to about 49, and most preferably about 30, bases in length. This fragment is preferably a fragment taken from between about xe2x88x922252 to about xe2x88x92607 nucleotide sequence of the ASA2 promoter.
A third aspect of the present invention is a DNA construct comprising an expression cassette, which construct comprises, in the 5xe2x80x2 to 3xe2x80x2 direction, an ASA2 promoter and a structural gene positioned downstream from the promoter and operatively associated therewith.
A fourth aspect of the present invention is an isolated DNA promoter sequence (included in SEQ ID NO: 14) derived by removing a portion of the ASA2 promoter, which is capable of directing high level constitutive transcription of a downstream structural gene in plant tissues. The promoter sequence may be selected from the group consisting of the tobacco ASA2 promoter and DNA sequences which are at least 70 percent homologous to a 606 or smaller fragment of the tobacco ASA2 promoter capable of directing constitutive expression.
A fifth aspect of the present invention is a DNA construct comprising an expression cassette, which construct comprises, in the 5xe2x80x2 to 3xe2x80x2 direction, the truncated ASA2 promoter (such as the promoter described in the second and fourth aspects of the present invention) and a structural gene positioned downstream from the promoter and operatively associated therewith. Also provided is the method for introducing such a construct into a cell, transforming the cell and expressing the structural gene in the transformed cell. Such a cell may be a plant cell which can be regenerated into a transformed plant which expresses the structural gene.
A sixth aspect of the present invention provides cultured cells and regenerated plants transformed by the constructs of the present invention. The transformed plant may be regenerated from the transformed plant cells.
A seventh aspect of the present invention provides for a method of selecting transformed plant cells which comprises the steps of: introducing into a plant cell an expression cassette comprising the ASA2 structural gene of the present invention which which encodes an AS which is substantially resistant to inhibition by free Trp or an amino acid analog of Trp to yield a transformed plant cell, and culturing the transformed plant cell in an amount of an amino acid analog of Trp, such as 5MT, that inhibits the growth of a corresponding plant cell which does not contain the ASA2 structural gene. This method can also be applied to cells of microorganisms, such as Escherichia coli (E. coli). In one embodiment of the invention, the expression cassette further contains a desirable gene. In another embodiment of the invention, the expression of the ASA2 structural gene is further inducible. In yet another embodiment of the invention, the desirable gene is under the control of a different promoter than that of the ASA2 structural gene; thus, the transformed cell or plant regenerated from the transformed cell can express the desirable gene independent of the expression of the ASA2 structural gene. The expression cassette, transformed cell and plant are also aspects of the invention.
An eighth aspect of the present invention provides for a method for imparting, to a plant cell, tolerance to an amino acid analog of Trp. The method comprises introducing an expression cassette containing the ASA2 structural gene of the present invention into cells of a wild-type plant to yield transformed plant cells, and expressing the ASA2 in an amount to render the transformed cells substantially tolerant to an amount of an amino acid analog of Trp that inhibits the growth of the untransformed cells of the wild-type plant. In one embodiment of the invention, the expression of the ASA2 structural gene is under the control of an inducible promoter. The transformed plant is also an aspect of the invention.
A ninth aspect of the present invention provides for altering the Trp content in a plant by transforming the plant cells with an expression cassette containing the ASA2 structural gene of the present invention, regenerating a differentiated plant from the transformed plant cells wherein the cells of the differentiated plant express ASA2 encoded by the expression cassette in an amount effective to increase the Trp content of the cells of the differentiated plant relative to the Trp content in the cells of the untransformed plant. In one embodiment of the invention, the expression of the ASA2 structural gene is controlled by an inducible promoter. The transformed plant is also an aspect of the invention.
A tenth aspect of the present invention provides for a method for producing AS which comprises the steps of: transforming a population of cells with expression cassettes comprising the ASA2 structural gene of the present invention, expressing the ASA2 in the cells, and recovering the expressed AS from the cells. The expressed AS may be further purified using methods known in the art with or without utilizing the antibodies of the present invention. The recombinant AS and substantially pure AS are also aspects of the present invention.
An eleventh aspect of the invention provides for the seeds (xe2x80x9ctransformed seedsxe2x80x9d) produced by any of the above transformed plants.
In the foregoing, ASA2 and its structural gene are merely used to illustrate the different aspects of the invention, one skilled in the art would realize that the foregoing is applicable to any AS and AS gene, and is preferably applicable to any AS and AS gene of the Nicotiana species (hereinafter referred to as xe2x80x9cNicotiana ASxe2x80x9d and xe2x80x9cNicotiana AS genexe2x80x9d, respectively).
The foregoing and other aspects of the present invention are explained in the discussion set forth-below.